Elucidating Hexanucleotide Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-Related SVA Retrotransposon in TAF1 with Nanopore Sequencing

Background: X-linked dystonia-parkinsonism (XDP) is an adult-onset neurodegenerative disorder characterized by progressive dystonia and parkinsonism. It is caused by a SINE-VNTR-Alu (SVA) retrotransposon insertion in the TAF1 gene with a polymorphic (CCCTCT)n domain that acts as a genetic modifier of disease onset and expressivity. Methods: Herein, we used Nanopore sequencing to investigate SVA genetic variability and methylation. We used blood-derived DNA from 96 XDP patients for amplicon-based deep Nanopore sequencing and validated it with fragment analysis which was performed using fluorescence-based PCR. To detect methylation from blood- and brain-derived DNA, we used a Cas9-targeted approach. Results: High concordance was observed for hexanucleotide repeat numbers detected with Nanopore sequencing and fragment analysis. Within the SVA locus, there was no difference in genetic variability other than variations of the repeat motif between patients. We detected high CpG methylation frequency (MF) of the SVA and flanking regions (mean MF = 0.94, SD = ±0.12). Our preliminary results suggest only subtle differences between the XDP patient and the control in predicted enhancer sites directly flanking the SVA locus. Conclusions: Nanopore sequencing can reliably detect SVA hexanucleotide repeat numbers, methylation and, lastly, variation in the repeat motif.

Clinical Performance of the cobas Liat SARS-CoV-2 & Influenza AB for the Detection of SARS-CoV-2 in Nasal Samples

Background and Objective: Point-of-care type molecular diagnostic tests have been used for detecting SARS-CoV-2, although their clinical utility with nasal samples has yet to be established. This study evaluated the clinical performance of the cobas Liat SARS-CoV-2 & Influenza AB (Liat) in nasal samples. Methods: Nasal and nasopharyngeal samples were collected and were tested using the Liat, the cobas 6800 system and the cobas SARS-CoV-2 & Influenza AB (cobas), and a method developed by National Institute of Infectious Diseases, Japan (NIID). Results: A total of 814 nasal samples were collected. The Liat assay was positive for SARS-CoV-2 in 113 (13.9%). The total, positive, and negative concordance rate between the Liat and cobas/NIID assays were 99.3%/98.4%, 99.1%/100%, and 99.3%/98.2%, respectively. Five samples were positive only using the Liat assay. Their Ct values ranged from 31.9 to 37.2. The Ct values of the Liat assay were significantly lower (p < 0.001) but were correlated (p < 0.001) with those of other molecular assays. In the participants who tested positive for SARS-CoV-2 on the Liat assay using nasopharyngeal samples, 88.2% of their nasal samples also tested positive using the Liat assay. Conclusion: The Liat assay showed high concordance with other molecular assays in nasal samples. Some discordance occurred in samples with Ct values > 30 on the Liat assay.

Analytical performances of the AMPLIQUICK® Respiratory Triplex assay for simultaneous detection and differentiation of SARS-CoV-2, influenza A/B and respiratory syncytial viruses in respiratory specimens

Although patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B and respiratory syncytial virus (RSV) show comparable or very similar manifestations, the therapeutic approaches of these respiratory viral infections are different, which requires an accurate diagnosis. Recently, the novel multiplex real-time reverse transcription-polymerase chain reaction assay AMPLIQUICK® Respiratory Triplex (BioSynex SA, Illkirch-Graffenstaden, France) allows simultaneous detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory tract samples. We herein evaluated the performance of the AMPLIQUICK® Respiratory Triplex for the detection of the four viruses in respiratory specimens, using Allplex™ Respiratory Panel 1 and 2019-nCoV assays (Seegene, Seoul, Korea) as reference comparator assays. A total of 359 archived predetermined respiratory samples, including 83, 145, 19 and 95 positive specimens for SARS-CoV-2, influenza A, influenza B and RSV respectively, were included. The AMPLIQUICK® Respiratory Triplex showed high concordance with the reference assays, with an overall agreement for SARS-CoV-2, influenza A, influenza B, and RSV at 97.6%, 98.8%, 98.3% and 100.0%, respectively, and high κ values ranging from 0.93 to 1.00, indicating an almost perfect agreement between assays. Furthermore, high correlations of cycle threshold (Ct) values were observed for positive samples of the four viruses between the AMPLIQUICK® Respiratory Triplex and comparator assays, with an overall high agreement between Ct values assessed by Bland-Altman analyses. In conclusion, these observations demonstrate that the multiplex AMPLIQUICK® Respiratory Triplex is a reliable assay for the qualitative detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory specimens, which may prove useful for streamlining diagnostics during the winter influenza-seasons.

Performance of HCV Antigen Testing for the Diagnosis and Monitoring of Antiviral Treatment: A Systematic Review and Meta-Analysis

Background and Aims. Active hepatitis C virus (HCV) infection is based on the detection of HCV RNA that it is effective but presents high cost and the need to hire trained personnel. This systematic review and meta-analysis is aimed at evaluating the diagnostic accuracy of HCV Ag testing to identify HCV cases and to monitor antiviral treatment including DAA treatment. Methods. The studies were identified through a search in PubMed, Lilacs, and Scopus from 1990 through March 31, 2020. Cohort, cross-sectional, and randomized controlled trials were included. Two independent reviewers extracted data and assessed quality using an adapted Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Our primary outcome was to determine the accuracy of HCV Ag detection for the diagnosis, which we estimated using random-effects meta-analysis. Results. Of 3,062 articles identified, 54 met our eligibility criteria. The studies described cohorts from 20 countries, including 14,286 individuals with chronic HCV individuals. Studies for ECLIA technology demonstrated highest quality compared to studies that used ELISA. The pooled sensitivity and specificity (95% CI) for HCV Ag detection of active HCV infection were 98.82% ( 95 % CI = 98.04 %; 99.30%) and 98.95% ( 95 % CI = 97.84 %; 99.49%), respectively. High concordance was found between HCV Ag testing and HCV RNA detection 89.7% and 95% to evaluate antiviral treatment. Conclusions. According to our findings, HCV Ag testing could be useful to identify HCV active cases in low-resource areas. For antiviral treatment, HCV Ag testing will be useful at the end of treatment.

Genotyping-in-Thousands by sequencing panel development and application to inform kokanee salmon (Oncorhynchus nerka) fisheries management at multiple scales

The ability to differentiate life history variants is vital for estimating fisheries management parameters, yet traditional survey methods can be inaccurate in mixed-stock fisheries. Such is the case for kokanee, the freshwater resident form of sockeye salmon (Oncorhynchus nerka), which exhibits various reproductive ecotypes (stream-, shore-, deep-spawning) that co-occur with each other and/or anadromous O. nerka in some systems across their pan-Pacific distribution. Here, we developed a multi-purpose Genotyping-in-Thousands by sequencing (GT-seq) panel of 288 targeted single nucleotide polymorphisms (SNPs) to enable accurate kokanee stock identification by geographic basin, migratory form, and reproductive ecotype across British Columbia, Canada. The GT-seq panel exhibited high self-assignment accuracy (93.3%) and perfect assignment of individuals not included in the baseline to their geographic basin, migratory form, and reproductive ecotype of origin. The GT-seq panel was subsequently applied to Wood Lake, a valuable mixed-stock fishery, revealing high concordance (>98%) with previous assignments to ecotype using microsatellites and TaqMan® SNP genotyping assays, while improving resolution, extending a long-term time-series, and demonstrating the scalability of this approach for this system and others.

The In Vivo Transcriptomic Blueprint of Mycobacterium tuberculosis in the Lung

Mycobacterium tuberculosis (Mtb) genes encoding proteins targeted by vaccines and drugs should be expressed in the lung, the main organ affected by Mtb, for these to be effective. However, the pulmonary expression of most Mtb genes and their proteins remains poorly characterized. The aim of this study is to fill this knowledge gap. We analyzed large scale transcriptomic datasets from specimens of Mtb-infected humans, TB-hypersusceptible (C3H/FeJ) and TB-resistant (C57BL/6J) mice and compared data to in vitro cultured Mtb gene-expression profiles. Results revealed high concordance in the most abundantly in vivo expressed genes between pulmonary Mtb transcriptomes from different datasets and different species. As expected, this contrasted with a lower correlation found with the highest expressed Mtb genes from in vitro datasets. Among the most consistently and highly in vivo expressed genes, 35 have not yet been explored as targets for vaccination or treatment. More than half of these genes are involved in protein synthesis or metabolic pathways. This first lung-oriented multi-study analysis of the in vivo expressed Mtb-transcriptome provides essential data that considerably increase our understanding of pulmonary TB infection biology, and identifies novel molecules for target-based TB-vaccine and drug development.

Equivalence of the GeneXpert System and GeneXpert Omni System for tuberculosis and rifampicin resistance detection

A laboratory validation study was conducted to assess the equivalence of Xpert MTB/RIF Ultra testing on the GeneXpert System and the GeneXpert Omni System (‘Omni’) for tuberculosis and rifampicin resistance. High concordance of the two devices was demonstrated for well-characterized clinical samples as well as control materials, with controls tested on Omni at normal and challenging environmental conditions (i.e. 35°C, 90% relative humidity). Equivalence of the Cts for all probes was also shown. Equivalence was demonstrated for the Omni and GeneXpert devices for tuberculosis and rifampicin resistance detection for a diverse range of clinical specimens and environmental conditions.